Limits...
Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse.

Volpert M, Mangum JE, Jamsai D, D'Sylva R, O'Bryan MK, McIntyre P - Sci Rep (2014)

Bottom Line: Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family.Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution).Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

View Article: PubMed Central - PubMed

Affiliation: Dept of Pharmacology, University of Melbourne, Parkville, VIC, Australia.

ABSTRACT
While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

Show MeSH

Related in: MedlinePlus

Mouse CRISP3 is incompletely digested by Endo H.(A) Mouse CRISP3 wild type, N99Q and N113Q are partially digested by Endo H (E). Complete digestion with PNGase F (P) is included for size comparison. (B) A size comparison of mouse and human CRISP3 (mCRISP3 and hCRISP3, respectively) glycans (square brackets). Full length gels are available under supplementary data; gels were run under the same conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3936225&req=5

f6: Mouse CRISP3 is incompletely digested by Endo H.(A) Mouse CRISP3 wild type, N99Q and N113Q are partially digested by Endo H (E). Complete digestion with PNGase F (P) is included for size comparison. (B) A size comparison of mouse and human CRISP3 (mCRISP3 and hCRISP3, respectively) glycans (square brackets). Full length gels are available under supplementary data; gels were run under the same conditions.

Mentions: N-glycosylation of human CRISP3 at N220 only was confirmed, with no observable glycosylation of hCRISP3-N220Q (Figure 6B, last lane), and no impairment in protein secretion.


Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse.

Volpert M, Mangum JE, Jamsai D, D'Sylva R, O'Bryan MK, McIntyre P - Sci Rep (2014)

Mouse CRISP3 is incompletely digested by Endo H.(A) Mouse CRISP3 wild type, N99Q and N113Q are partially digested by Endo H (E). Complete digestion with PNGase F (P) is included for size comparison. (B) A size comparison of mouse and human CRISP3 (mCRISP3 and hCRISP3, respectively) glycans (square brackets). Full length gels are available under supplementary data; gels were run under the same conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3936225&req=5

f6: Mouse CRISP3 is incompletely digested by Endo H.(A) Mouse CRISP3 wild type, N99Q and N113Q are partially digested by Endo H (E). Complete digestion with PNGase F (P) is included for size comparison. (B) A size comparison of mouse and human CRISP3 (mCRISP3 and hCRISP3, respectively) glycans (square brackets). Full length gels are available under supplementary data; gels were run under the same conditions.
Mentions: N-glycosylation of human CRISP3 at N220 only was confirmed, with no observable glycosylation of hCRISP3-N220Q (Figure 6B, last lane), and no impairment in protein secretion.

Bottom Line: Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family.Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution).Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

View Article: PubMed Central - PubMed

Affiliation: Dept of Pharmacology, University of Melbourne, Parkville, VIC, Australia.

ABSTRACT
While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

Show MeSH
Related in: MedlinePlus